Human skin is made up mainly of two main layers, namely the dermis and the epidermis that superficially covers the dermis. The dermis provides the epidermis with a solid support. It is also its nourishing element. It is made up mainly of fibroblasts and an extracellular matrix composed mainly of collagen, elastin and a substance known as ground substance. These components are synthesized by the fibroblasts.
The cohesion between the epidermis and the dermis is provided by the dermo-epidermal junction. This is a complex region about 100 nm thick, which comprises the basal pole of the basal keratinocytes, the epidermal membrane and the sub-basal zone of the superficial dermis. From a structural viewpoint, hemidesmosomes, into which are inserted keratin filaments (hemidesmosome-tonofilament complex) are distributed on the plasma membrane of the basal keratinocytes. Facing these hemidesmosome-tonofilament complexes are anchoring filaments that cross the epidermal basal membrane. The anchoring filaments are attached to laminin V on the epidermal side. It has been shown that these anchoring fibrils, which are entirely visible by electron microscopy, are composed of type VII collagen (referred to as collagen VII hereinbelow). The collagen VII is synthesized by keratinocytes and fibroblasts, but more substantially by keratinocytes (Aumailley M., Rousselle P. Laminins of the dermoepidermal junction. Matrix Biology, 1999, 18: 19-28; Nievers M., Schaapveld R., Sonnenberg A. Biology and function of hemidesmosomes. Matrix Biology, 1999, 18: 5-17).
Collagens are the major proteins of the extracellular matrices of the skin. To date, 20 types of collagen have been identified, and are noted from I to XX. Collagens are not all synthesized by the same cell types, regulation of their expression differs from one collagen to another, and all collagen molecules are variants of a common precursor, which is the α chain of procollagen.
The dermoepidermal junction is a structure that conditions the surface state of the skin. Thus, a dermoepidermal junction with intact anchoring structures is maintained folded, thus making it possible to increase the surface area of the contact zone between the dermis and the epidermis, to promote exchanges of diffusible factors, especially between these two tissues, to reinforce their cohesion and to improve the appearance of the epidermis. In cases where the anchoring structures are impaired, in particular due to a deficiency in the synthesis of collagen IV, collagen VII or laminin V and/or due to aging, this causes flattening of the dermoepidermal junction. Fewer exchanges take place, the two tissues are less solidly connected, the epidermis folds, and, as the skin is less firm and less taut, wrinkles appear and the fragility of the skin with respect to mechanical attack is increased.
With age, collagen becomes thinner and disorganised, the renewal of the skin cells decreases, wrinkles appear on the surface of the skin, and the skin is duller and less firm. Cutaneous aging is conditioned by genetic factors. Moreover, certain environmental factors such as smoking and especially exposure to sunlight accelerate it. The skin thus has a much more aged appearance on the areas exposed to sunlight, such as the back of the hands or the face. Thus, these other factors also have a negative impact on the natural collagen of the skin.
Consequently, given the important role of collagen in the integrity of the skin and in its resistance to external attacking factors of mechanical type, stimulation of the synthesis of these collagens, and in particular of procollagen I, collagen IV, collagen VII and laminin V, appears to be an effective means for overcoming the signs of aging of the skin.
To overcome the abovementioned drawbacks, to improve the appearance of the skin, to improve its mechanical properties and to avoid the pathologies associated with cell deficiency, deficiency of cell renewal or deficiency of certain compounds of the dermis or of the dermoepidermal junction, the inventors view it to be important to develop products that are aimed at reinforcing or maintaining the role as a support and a nourishing factor played by the dermis, the cohesion between the various layers of the skin, and more particularly the cohesion between the dermis and the epidermis, by increasing keratinocyte proliferation, by stimulating fibroblast proliferation and metabolism and by stimulating the synthesis of collagens, in particular of procollagen I, collagen IV, collagen VII and laminin V.
The epidermis, which covers the dermis and is in direct contact with the external environment, has the main role of protecting the body against the dehydration and external attack. Natural human epidermis is composed mainly of three types of cell, namely keratinocytes, which form the vast majority, melanocytes and Langerhans cells. Each of these cell types contributes, by virtue of its intrinsic functions, towards the essential role played in the body by the skin.
The dermis is made up mainly of fibroblasts and an extracellular matrix. Leucocytes, mastocytes and tissue macrophages are also found therein. It is also made up of blood vessels and nerve fibres.
The extracellular matrix of the dermis, like that of all the connective tissues of the body, is composed of proteins belonging to several major families: collagens, matrix glycoproteins other than collagens (fibronectin, laminin), elastin and proteoglycans. Glycosaminoglycans in free form (i.e. not bound to a protein) are also found in the extracellular matrix of the dermis, as in all connective tissues of the body.
It is now well established that specific interactions exist between these various classes of proteins to give rise to a functional tissue.
Proteoglycans are complex macromolecules formed from a branched central protein trunk, or protein network, to which are attached numerous polysaccharide side chains known as glycosaminoglycans.
In the rest of the present patent application, proteoglycans will be denoted by the abbreviation PG and glycosaminoglycans by the abbreviation GAG.
GAGs were for a long time referred to as acidic mucopolysaccharides on account of their high water-retaining capacity, their carbohydrate nature and their acidic nature originating from their multiple negative charges.
Thus, the polarity of GAGs makes them implicitly participate in certain biological functions such as the hydration of tissues, the binding of cations or the barrier role of ionic filtration.
PGs and GAGs are synthesized by various cells in the dermis and the epidermis: fibroblasts, keratinocytes and melanocytes.
The fibroblasts mainly synthesize collagens, matrix glycoproteins other than collagens (fibronectin, laminin), proteoglycans and elastin. The keratinocytes mainly synthesize sulfated GAGs and hyaluronic acid, while the melanocytes apparently do not produce any hyaluronic acid.
When they are incorporated in a PG, GAGs are linear chains composed of a repetition of a base diholoside always containing a hexosamine (glucosamine or galactosamine) and another saccharide (glucuronic acid, iduronic acid or galactose). The glucosamine is either N-sulfated or N-acetylated. On the other hand, the galactosamine is always N-acetylated. In addition, there may be sulfates O-bonded to the hexosamine, uronic acid and galactose.
The strong anionic nature of GAGs is explained by the presence of carboxylate groups in the hexuronic acids (glucuronic acid and iduronic acid) and of O- and N-bonded sulfate groups.
The main GAGs are hyaluronic acid or hyaluronan (HA), heparan sulfate (HS), heparin (HP), chondroitin, chondroitin sulfate (CS), chondroitin 4-sulfate or chondroitin sulfate A (CSA), chondroitin 6-sulfate or chondroitin sulfate C (CSC), dermatan sulfate or chondroitin sulfate B (CSB) and keratan sulfate (KS), which differs from the other glycosaminoglycans by the presence of galactose instead of uronic acid.
When they are combined with a protein in the form of PG, the GAGs are linked via anchoring structures to the various polypeptide chains, named the “core” protein or carrier protein, and thus form PG molecules.
GAGs may also exist in the extracellular matrix in free form, ie not bound to a matrix protein: this is especially the case for hyaluronic acid.
During the synthesis of PGs, the GAGs are polymerized from these anchoring structures.
The synthesis of GAGs requires the coordinated and concerted action of very specific enzymes (transferases, epimerases and sulfotransferases) that are adjacent in the membrane of the endoplasmic reticulum and of the golgi bodies. Next, a multitude of biochemical reactions (N-deacetylation, N- and O-sulfation, and epimerization) modify the two constituent saccharides of the base unit, heterogeneously along the chain. For example, from one heparan sulfate chain to another, the glucuronic acid/iduronic acid ratio, the nature, number and position of the O-sulfations, and the N-sulfate/O-sulfate ratio may vary, which essentially offers immense structural diversity.
In general, the biological roles of PGs are highly diversified, ranging from a passive mechanical support function (for example serglycines) or an ionic barrier role in molecular filtration (for example perlecane and bamacane of the glomerular basal membrane), to more specific effects in cell adhesion, spreading, proliferation and differentiation or morphogenesis, or to highly specific effects of PG-protein interactions, such as the beta-glycan receptor function or the interaction of decorin with collagen.
One of the roles of dermal connective tissue is to protect the body against external attack when simultaneously forming an informative interface.
To do this, the dermis has high mechanical strength while maintaining, however, great flexibility.
Its strength is ensured by the dense network of collagen fibres, but it is the PGs and the hyaluronic acid which, by ensuring the moisturization, distribution and suppleness of the fibres, make the difference between the skin and, for example, leather.
The PGs constitute 0.5% to 2% of the dry weight of the dermis, collagen alone representing up to 80% of this weight.
The concentration and distribution in human skin of GAGs and PGs vary with age.
Hyaluronic acid or hyaluronan (HA) is the main GAG of the dermis, the latter containing half the HA of the body.
The synthesis of HA is performed especially by the fibroblasts, close to the inner face of the plasma membrane. It is performed continuously. This gigantic polysaccharide (several million daltons) has a very high intrinsic viscosity, ensuring the moisturization and assembly of the various components of the connective tissue by forming supramolecular complexes.
Dermatan sulfate (DS), which was first isolated from the dermis, is also widely abundant in the skin. It constitutes 40% to 50% of the dermal GAGs.
In parallel with the mechanisms contributing to the development of these specialized extracellular matrices, continuous remodeling processes exist, the regulation of which depends on the balance between the synthesis and degradation of the protein components of the matrix.
Several families of matrix proteases are now described, as are the factors involved in their activation-inactivation.
In the course of chronological and/or actinic aging, the dermis and the epidermis undergo several changes and degradations which are reflected, with age, by flaccidness and a loss of suppleness of the skin.
Among the components degraded (especially collagen and elastin), the PGs and GAGs are also adversely affected. Specifically, over the course of aging, the fibroblasts and keratinocytes produce less and less PGs and GAGs and their synthesis is imperfect. This results in considerable disorganization: the deposition of GAGs on the protein skeleton forming the PG is abnormal, the consequence of this is a reduced avidity for water of these PGs thus a reduction in the moisturization and tonicity of tissues.
Restoring a normal production of PGs and GAGs by fibroblasts and keratinocytes contributes partially towards compensating for the loss of moisturization of the skin.
The degradation of these matrices thus contributes towards the phenomenon of dryness and of loss of suppleness of the skin.
The importance of having available products whose effects are directed towards maintaining the level of PGs and GAGs in the skin and thus of maintaining, inter alia, good moisturization and good suppleness of the skin, effectively combating the signs of aging, may thus be appreciated.